CN116284370A - Multimeric glycosylated hemoglobin monoclonal antibody and preparation method thereof - Google Patents
Multimeric glycosylated hemoglobin monoclonal antibody and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the technical field of glycosylated hemoglobin, in particular to a multimeric glycosylated hemoglobin monoclonal antibody and a preparation method thereof, wherein the sequence of the monoclonal antibody is shown as SEQ ID NO:1, the sequences of the light chain variable region and the heavy chain variable region are respectively shown in SEQ ID NO: 2-3. The preparation method comprises the following steps: 1) Immunizing an animal; 2) Cell fusion; 3) Selectively culturing; 4) Screening and cloning hybridoma positive clones; 5) Purifying monoclonal antibodies; 6) The monoclonal antibodies are cross-linked. The multimeric monoclonal antibody prepared by the method can be used for preparing a standard substance, has strong specificity, can obtain high-purity antibodies from the immunogen with impure requirements on the immunogen, is easy to standardize, has small difference between batches, can identify and detect only one epitope on the antigen, is difficult to cross react, can not cause cross reaction precipitation and agglutination reaction with other proteins, and is favorable for result comparison and quality control of various factories and laboratories.
Description
Technical Field
The invention relates to the technical field of glycosylated hemoglobin, in particular to a multimeric glycosylated hemoglobin monoclonal antibody and a preparation method thereof.
Background
Glycosylated hemoglobin is a gold standard for measuring glycemic control and is also an important means for diagnosing and managing diabetes. In the treatment of diabetes, glycosylated hemoglobin levels are of clinical importance in assessing overall control of blood glucose, in finding problems in the treatment, and in guiding the treatment regimen.
Normal people have three haemoglobins: hbA, hbF, hbA2, human erythrocytes mainly contain HbA. When hemoglobin is separated by chromatography, 3 sugar-containing components can be eluted, namely: hbA1a, hbA1b, hbA1c are collectively referred to as glycosylated hemoglobin. HbA1c is glycosylated hemoglobin of glucose, and the other two are glycosylated hemoglobin of other sugars.
Glycosylated hemoglobin is formed by HbA binding glucose during metabolism, so that it accurately reflects the glucose level in blood. Glycosylated hemoglobin is the product of the binding of hemoglobin in erythrocytes in human blood to blood glucose. The combination of blood glucose and hemoglobin produces glycosylated hemoglobin, which is an irreversible reaction and is proportional to the blood glucose concentration, and is maintained for about 120 days, so that the blood glucose concentration before 120 days can be observed. Glycosylated hemoglobin tests can generally reflect blood glucose control in patients for approximately 8-12 weeks.
The principle of the immunoassay of glycosylated hemoglobin is that a specific antibody capable of recognizing a plurality of glycosylated amino acids at the amino terminal of the beta-chain of glycosylated hemoglobin is reacted with HbA1c to form an antigen-antibody complex for detection. The method is quick and simple, and can be detected on an automatic instrument.
Disclosure of Invention
The invention aims to provide a multimeric glycosylated hemoglobin monoclonal antibody and a preparation method thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a multimeric glycosylated hemoglobin monoclonal antibody having the sequence of SEQ ID NO: 1. Wherein, the light chain variable region sequence is shown as SEQ ID NO: 2. The heavy chain variable region sequence is shown in SEQ ID NO: 3.
The preparation method of the multimeric glycosylated hemoglobin monoclonal antibody comprises the following steps:
(1) Immunization of animals
Selecting female BALB/c mice of 6-8 weeks old, injecting the antigen into peripheral immune organs through blood circulation or lymph circulation, stimulating corresponding B lymphocyte clone, activating and proliferating the B lymphocyte clone, and differentiating the B lymphocyte clone into sensitized B lymphocyte;
(2) Cell fusion
By CO 2 Killing mice by gas, taking out spleens by aseptic operation, extruding and grinding in a plate, and preparing spleen cell suspension; mixing the prepared syngeneic myeloma cells with the spleen cells of the mice according to a certain proportion, and adding a fusogenic agent polyethylene glycol; under the action of polyethylene glycol, various lymphocytes are fused with myeloma cells to form hybridoma cells;
(3) Selective cultivation
Screening the fused hybridoma cells by adopting a HAT selective medium;
(4) Screening and cloning of hybridoma-positive clones
Cloning and culturing the hybridoma cells by adopting a limiting dilution method; screening out positive hybridoma cells capable of generating the required monoclonal antibody, and carrying out cloning amplification; through comprehensively identifying the type, subclass, specificity and affinity of the immunoglobulin of the secreted monoclonal antibody, identifying the epitope of the antigen and the molecular weight of the epitope, the monoclonal antibody is frozen in time;
(5) Monoclonal antibody purification
Purifying the ascites of the mice by using an ammonium sulfate salting-out method and a G protein chromatography method;
(6) The monoclonal antibodies are cross-linked.
Wherein, the step (1) specifically comprises the following steps:
primary immunization, 50 ug/Ag, is injected subcutaneously with Freund's complete adjuvant in multiple spots, typically 1.5mL, 3 weeks apart;
the second immunization, the dose route is the same as above, and Freund's incomplete adjuvant is added at intervals of 3 weeks;
the third immunization, the dose is the same, no adjuvant is added, the intraperitoneal injection is carried out, the blood is taken after 7 days to measure the titer, and the immunization effect is detected at intervals of 3 weeks;
boosting, dosage of 50 mug, intraperitoneal injection; after 3 days, spleen was taken for fusion.
Wherein, the step (2) specifically comprises:
1) Preparation of feeder cells
BALB/c mice aged 6-10 weeks; pulling neck, killing, soaking in 75% alcohol, sterilizing for 3min, shearing skin with sterile scissors, and exposing peritoneum; injecting 6mL of culture solution into the tube, repeatedly flushing, and sucking out flushing liquid; putting into a 10mL centrifuge tube, and centrifuging at 1200rpm for 5min;
suspending with culture medium of 20% calf serum, and adjusting cell number to 1×10 5 /mL; add 96-well plates, 100 μl/well; culturing in incubator at 37deg.C;
2) Preparation of myeloma cells
Expanding and culturing myeloma cells 48-36 hours before fusion; on the day of fusion, lightly blowing down cells from the bottle wall by using an elbow dropper, and collecting the cells in a 50m centrifuge tube or a fusion tube; centrifuging at 1000r/min for 5-10 min, and discarding supernatant; adding 30mL of incomplete culture medium, and centrifugally washing once; then, re-suspending the cells in 10mL of incomplete culture medium, and uniformly mixing; taking myeloma cell suspension, adding 0.4% of a phenol blue dye solution as living cells for counting for later use;
3) Preparation of spleen cells
Taking an immunized BALB/c mouse, removing eyeballs, taking blood, and separating serum to be used as positive control serum in antibody detection; meanwhile, killing the mice through cervical dislocation, soaking in 75% alcohol for 5 minutes, lifting the skin at the left side abdomen after fixing on a culture dish, seeing spleen, changing ophthalmic scissors forceps, cutting peritoneum in an ultra clean bench by aseptic operation, taking out the spleen, placing the spleen in a dish which is filled with 10mL of incomplete culture medium, lightly washing, and carefully stripping off surrounding connective tissues; stainless steel screen in plateOn the net, grinding the needle core of the injector into cell suspension, counting, and enabling spleen cells to enter an incomplete culture medium in a plate; blowing with a suction tube for several times to prepare single cell suspension; typically 1X 10 mice per mouse 8 -2.5×10 8 Individual spleen cells;
4) Cell fusion
Will be 1X 10 8 Spleen cells and 1X 10 7 Myeloma cell SP2/0 is mixed in a 50mL fusion tube, and incomplete culture medium is added to 30mL, and fully and uniformly mixed; centrifuging at 1000r/min for 5-10 min, and sucking the supernatant as much as possible; tapping the fusion tube bottom on the palm to loosen and uniformly deposit cells;
1mL of preheated 50% PEG was added over 30s with a 1mL pipette with gentle stirring; sucking the suction tube and standing for 1min; adding the preheated incomplete culture solution, stopping PEG effect, and continuously adding 1mL,2mL,3mL,4mL,5mL and 10mL in every 2 min; centrifuging at 800rpm for 5 minutes; discarding the supernatant;
adding 5mL of complete culture medium, gently blowing and sucking the precipitated cells, suspending and uniformly mixing the cells, and then supplementing the complete culture medium to 40-50mL; split charging 96-well cell culture plates, 100 μl per well, then placing the plates at 37deg.C, 5% CO 2 Culturing in an incubator; supplementing a selection medium after 6 hours; 50 mu L of each well, and half-changing the liquid with a selective medium after 3 days;
observing the growth condition of the hybridoma cells, and sucking out the supernatant for antibody detection when the hybridoma cells grow to more than 1/10 of the bottom area of the hole.
Wherein, the step (3) is concretely that the antigen is diluted to 10ug/mL by coating liquid; adding 100 mu L/hole into the enzyme-labeled plate hole, and standing at 4 ℃ overnight or at 37 ℃ for adsorption for 2 hours; discarding the liquid in the hole, washing 3 times with washing liquid for 3 minutes each time, and drying; 100 mu L of sealing liquid is added to each hole to seal for 1 hour at 37 ℃; washing 3 times with washing liquid;
adding 100 mu L of hybridoma cell culture supernatant to be detected into each hole, and simultaneously setting positive control, negative control and blank control; incubation at 37 ℃ for 1 hour; washing and drying;
adding enzyme-labeled secondary antibody, incubating for 1 hour at 37 ℃ at 100 mu L per hole, washing, and drying;
adding substrate liquid, adding 100 mu L of freshly prepared substrate use liquid into each hole, and 20 minutes at 37 ℃;
in a ratio of 2mol/L H 2 SO 4 Terminating the reaction, and reading an OD value on an ELISA reader;
and (3) result judgment: P/N is equal to or greater than 2.1, or P is equal to or greater than N+3SD; if the negative control Kong Mose is or is near colorless, the positive control wells are clearly colored, and the results can be directly observed with the naked eye.
Wherein, the step (4) is specifically that the spleen cells of the mice are prepared as feeder cells; preparing hybridoma cell suspension to be cloned, and diluting the hybridoma cell suspension to 3 different dilutions of 5, 10 and 20 cells per milliliter by using HT medium containing 20% serum;
added according to 5X 10 per milliliter 4 -1×10 5 Proportion of cells, respectively adding abdominal macrophages into the hybridoma cell suspension; split charging each hybridoma cell into one 96-well plate, wherein the amount of each well is 100 mu L;
37℃、5％ CO 2 culturing for 6 days, and detecting antibodies after macroscopic cloning; observing under an inverted microscope, marking a hole in which only a single clone grows, and taking supernatant as an antibody for detection;
and (5) taking cells of the antibody detection positive hole, performing expansion culture, and freezing.
Wherein, the step (5) is specifically that an affinity purification method is adopted, staphylococcus protein A or an anti-mouse globulin antibody is used for crosslinking with a carrier, and an affinity chromatographic column is prepared for eluting after the antibody is combined.
Wherein, in the step (6), the reaction system for coupling glycosylated hemoglobin monoclonal antibody is as follows:
antibody, concentration 2mg/mL,5mL;
glutaraldehyde, mass concentration 0.25%,200 μl;
centrifuging at 200rpm, and at 25 ℃ for 5 hours;
5mg/mL sodium borohydride was terminated.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides specific materials by adopting hybridoma technology to prepare glycosylated hemoglobin immune latex in a turbidimetric manner, the prepared multimeric glycosylated hemoglobin monoclonal antibody has stronger specificity, and the results of ascites titer determination and competitive ELISA method tests show that the HbA1c multimeric monoclonal antibody prepared and screened by the invention has better specificity and sensitivity.
(2) The multimeric monoclonal antibody prepared by the method can be used for preparing a standard substance, has strong specificity, can obtain high-purity antibodies from the immunogen with impure requirements on the immunogen, is easy to standardize, has small difference between batches, can identify and detect only one epitope on the antigen, is difficult to cross react, can not cause cross reaction precipitation and agglutination reaction with other proteins, and is favorable for result comparison and quality control of various factories and laboratories.
(3) The monoclonal antibody prepared by the method can also be used as a coated multimeric monoclonal antibody of an enzyme-linked immunosorbent assay (ELISA) to detect antigen titer.
Drawings
FIG. 1 is a flow chart of the preparation of the monoclonal antibody of the present invention.
FIG. 2 is a SDS-PAGE electrophoresis; wherein M: protein marker;1: cracking sample; 2-4: flowing out the sample; 5-13: cleaning a sample; 14: eluting the sample.
Fig. 3 is a calibration graph.
Fig. 4 is a linear relationship diagram.
FIG. 5 shows the results of stability analysis.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIG. 1, a method for preparing a multimeric glycosylated hemoglobin monoclonal antibody comprises the following steps:
(1) Immunization of animals
Immunization of an animal is the process of immunizing a mouse with an antigen of interest, causing the mouse to produce sensitized B lymphocytes. Female BALB/c mice of 6-8 weeks of age are generally selected for immunization according to a pre-established immunization schedule. The antigen enters peripheral immune organs through blood circulation or lymphatic circulation, stimulates corresponding B lymphocyte clones, activates, proliferates and differentiates into sensitized B lymphocytes.
The specific operation steps are as follows:
the immunization regimen is selected based on the nature of the antigen, and adjuvants are generally added to the soluble antigen which are poorly immunogenic. Common adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant are required to be mixed together in equal volumes and ground into a water-in-oil chylomorphic form.
Primary immunization, ag50 ug/mouse, was given by subcutaneous multipoint injection with Freund's complete adjuvant, typically 1.5mL, 3 weeks apart.
The second immunization, the dose route was the same as above, with incomplete Freund's adjuvant added at 3 weeks intervals.
The third immunization, the dose is the same, no adjuvant is added, the intraperitoneal injection is carried out, the blood is taken after 7 days to measure the titer, and the immunization effect is detected at intervals of 3 weeks. Boosting, 50ug of dosage, and intraperitoneal injection. After 3 days, spleen was taken for fusion.
(2) Cell fusion
By CO 2 Mice were sacrificed by air, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in plates. Mixing the prepared syngeneic myeloma cells with the spleen cells of the mice according to a certain proportion, and adding a fusogenic agent polyethylene glycol. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells.
The specific operation steps are as follows:
1) Preparation of feeder cells
During the selective culture process after cell fusion, since a large number of myeloma cells and spleen cells die successively, single or few dispersed hybridoma cells are likely to be difficult to survive, and other living cells must be added to propagate, and such added living cells are called feeder cells.
Preparation of mouse peritoneal macrophages:
BALB/c mice of 6-10 weeks old were sacrificed by pulling the neck, soaked in 75% alcohol, sterilized for 3min, and the skin was cut off with sterile scissors to expose the peritoneum. 6mL of the culture solution was poured into the flask with a pipette, and the flask was repeatedly rinsed and the rinse solution was aspirated. Put into a 10mL centrifuge tube and centrifuged at 1200rpm for 5min.
Suspending with culture medium of 20% calf serum, and adjusting cell number to 1×10 5 /mL; 96-well plates, 100. Mu.L/well, were added. Culturing in incubator at 37deg.C.
2) Preparation of myeloma cells
Myeloma cells were expanded 48-36 hours prior to fusion. On the day of fusion, cells were gently blown down from the walls of the flask with an elbow dropper and collected in a 50mL centrifuge tube or fusion tube.
Centrifuging at 1000r/min for 5-10 min, and discarding supernatant.
30mL of the incomplete medium was added, and the mixture was washed once by centrifugation. The cells were then resuspended in 10mL of incomplete medium and mixed well.
Taking myeloma cell suspension, adding 0.4% of a phenol blue dye solution as living cells for counting for later use.
3) Preparation of spleen cells
BALB/c mice that had been immunized were taken, the eyeballs were removed to collect blood, and serum was isolated as a positive control serum for antibody detection. Meanwhile, killing the mice through cervical dislocation, soaking in 75% alcohol for 5 minutes, fixing on a culture dish, lifting the skin at the left side abdomen, changing the forceps of the eye, cutting off the peritoneum in an ultra clean bench by aseptic operation, taking out the spleen, placing the spleen in a dish which is filled with 10mL of incomplete culture medium, slightly washing, and carefully stripping off surrounding connective tissues. The cells were placed on a stainless steel screen in a plate, and the cell suspension was ground with a syringe needle and counted. Spleen cells were allowed to enter the incomplete medium in the dish. The single cell suspension is prepared by blowing with a suction tube for several times. Typically 1X 10 mice per mouse 8 -2.5×10 8 Spleen cells.
4) Cell fusion
Will be 1X 10 8 Spleen cells and 1X 10 7 Myeloma cell SP2/0 is mixed in a 50mL fusion tube, and the incomplete culture medium is added to 30mL and fully mixed。
Centrifuging at 1000r/min for 5-10 min, and sucking the supernatant as much as possible.
Tapping the fusion tube bottom on the palm to loosen and uniformly deposit cells;
1mL of preheated 50% PEG was added over 30s using a 1mL pipette with gentle agitation.
The suction tube was allowed to stand for 1min.
The preheated incomplete culture solution is added to stop PEG action, and 1mL,2mL,3mL,4mL,5mL and 10mL are added continuously every 2 min. 800rpm,5 minutes; the supernatant was discarded.
5mL of complete medium is added, the precipitated cells are gently blown and sucked, suspended and mixed evenly, and then the complete medium is added to 40-50mL. Split charging 96-well cell culture plates, 100 μl per well, then placing the plates at 37deg.C, 5% CO 2 Culturing in an incubator.
After 6h, the selection medium was supplemented. 50 μl per well. Half-changing with selective medium after 3 days.
Hybridoma cell growth is often observed, and when the hybridoma cell grows to more than 1/10 of the hole bottom area, the supernatant is sucked out for antibody detection.
(3) Selective cultivation
The goal of the selective culture is to screen for fused hybridoma cells, typically using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but do not survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine-guanine-phosphoribosyl transferase obtained from spleen cells and the unlimited proliferation of myeloma cells.
The specific operation steps are as follows:
the antigen was diluted to 10ug/mL with coating solution.
The mixture was added to the wells of the enzyme-labeled plate at a concentration of 100. Mu.L/well, and the mixture was allowed to stand at 4℃overnight or at 37℃for adsorption for 2 hours.
The liquid in the wells was discarded and simultaneously washed 3 times with wash liquid for 3 minutes each time, and the wells were dried by patting.
100 mu L of sealing liquid is added to each hole to seal for 1 hour at 37 ℃;
washing 3 times with washing liquid;
adding 100 mu L of hybridoma cell culture supernatant to be detected into each hole, and simultaneously setting positive control, negative control and blank control; incubation at 37 ℃ for 1 hour; washing and drying.
The enzyme-labeled secondary antibody was added and incubated at 37℃for 1 hour at 100. Mu.L per well, washed and dried by pipetting.
The substrate solution was added, and 100. Mu.L of the freshly prepared substrate use solution was added to each well for 20 minutes at 37 ℃.
The reaction was stopped at 2mol/L H2SO4 and the OD was read on an ELISA reader.
And (3) result judgment: P/N.gtoreq.2.1, or P.gtoreq.N+3SD. If the negative control Kong Mose is or is near colorless, the positive control wells are clearly colored, and the results can be directly observed with the naked eye.
(4) Screening and cloning of hybridoma-positive clones
The hybridoma cells grown in HAT medium are only a few that secrete monoclonal antibodies of predetermined specificity, and therefore, must be selected and cloned. Cloning culture of hybridoma cells is generally performed by limiting dilution. Positive hybridoma cells which can produce the required monoclonal antibodies are screened out by adopting a sensitive, rapid and specific immunological method, and are subjected to cloning amplification. Through comprehensive identification of the type, subclass, specificity, affinity and antigen-recognizing epitope and its molecular weight of the secreted monoclonal antibody, the monoclonal antibody is frozen in time.
The specific operation steps are as follows:
mouse spleen cells were prepared as feeder cells.
Hybridoma cell suspensions to be cloned were prepared and diluted with HT medium containing 20% serum to 3 different dilutions of 5, 10 and 20 cells per ml. Added according to 5X 10 per milliliter 4 -1×10 5 Proportion of cells to the hybridoma cell suspensions, peritoneal macrophages were added, respectively.
Each of which is a solidHybridoma cells were dispensed in 96-well plates at a volume of 100 μl per well. 37 ℃ and 5% CO 2 Culturing for 6 days, and detecting antibodies after macroscopic cloning; under an inverted microscope, wells were identified in which only a single clone grew, and supernatants were used for antibody detection.
And (5) taking cells of the antibody detection positive hole, performing expansion culture, and freezing.
(5) Monoclonal antibody purification
The purification method of the monoclonal antibody is the same as that of the polyclonal antibody, the concentration of the ascites specific antibody is higher than that of the polyclonal antibody in the antiserum, and the purification effect is good. The corresponding purification method is adopted according to the required purity. Generally, the purification purpose is achieved by salting out, gel filtration, ion exchange chromatography and other steps, and a simpler acid precipitation method is also adopted. The most effective monoclonal antibody purification method at present is an affinity purification method, which adopts staphylococcus protein A or anti-mouse globulin antibody to crosslink with a carrier (commonly used Sepharose) to prepare an affinity chromatographic column to combine the antibody and then elute, and the recovery rate can reach more than 90 percent. Proteins can bind to IgG1, igG2a, igG2b, and IgG3, while also binding to small amounts of IgM. The concentration of the antibody in the eluate was roughly measured by ultraviolet light absorption, and 1.44 (absorbance unit) was 1mg/mL at A280nm of the mouse IgG monoclonal antibody solution. The pre-placement of the neutralization solution or rapid addition of the neutralization solution in the collection tube after low pH elution is critical to maintaining the activity of the purified antibodies. SDS-PAGE electrophoresis is shown in FIG. 2.
The antibody sequence is shown in SEQ ID NO: 1. Wherein, the light chain variable region sequence is shown as SEQ ID NO: 2. The heavy chain variable region sequence is shown in SEQ ID NO: 3.
(6) Monoclonal antibody cross-linking
One type of chemical crosslinking agent has a stable crosslinking bridge after crosslinking, and is generally non-cleavable. Glutaraldehyde is commonly used as such a reagent. Such agents have two reactive aldehyde groups that can react with amino groups on side chains of amino acid residues in the protein molecule to form crosslinks.
Glycosylated hemoglobin monoclonal antibody coupling reaction system:
antibody, concentration 2mg/mL,5mL;
glutaraldehyde (0.25%), 200. Mu.L
200rpm，25℃，5h；
Sodium borohydride stopped (5 mg/mL).
The performance of the product obtained by separating and purifying the method is tested, and the method is concretely as follows:
(1) Calibration curve
The total hemoglobin and glycosylated hemoglobin in the sample are immobilized by the same nonspecific adsorption with the latex microsphere, and when the specific monoclonal antibody is added, a latex-glycosylated hemoglobin-mouse anti-human HbA1C polyclonal monoclonal antibody complex is formed, the complex forms aggregation due to the goat anti-mouse IgG antibody, and the aggregation amount is different according to the immobilized glycosylated hemoglobin on the surface of the latex microsphere. The percentage of HbA1C in the sample can be determined by measuring the absorbance and comparing it with a standard curve of HbA1C percentage concentration. Linear range: 2-14% and a correlation coefficient R2>0.99, as shown in FIG. 3.
(2) Linear relationship
The HbA1C high-value whole blood sample is diluted by multiple times, and the self-made HbA1C latex turbidimetric reagent is used for detection, so that the correlation between the measured sample concentration and the diluted concentration is 0.99, as shown in FIG. 4.
(3) Precision of
The low-value and high-value HbA1C whole blood samples are respectively measured for 10 times in parallel by using a latex turbidimetric reagent, and the variation coefficient CV is within 3 percent, which indicates that the polyclonal monoclonal antibody can be used for the latex turbidimetric reagent and has good precision. The results are shown in Table 1.
TABLE 1
(4) Stability analysis
HbA1C monoclonal antibodies are respectively placed at-20 ℃, 2-8 ℃ and 37 ℃ and are prepared into latex reagents after 3-4 days, hbA1C calibrator with different concentrations is detected, the results are shown in figure 5, and the results show that the antibody has good stability at 37 ℃.
The experimental results show that the multimeric glycosylated hemoglobin monoclonal antibody prepared by the invention has the characteristics of high purity, high correlation, good stability, good precision, stability analysis and the like.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A multimeric glycosylated hemoglobin monoclonal antibody, characterized in that: the sequence of the polypeptide is shown in SEQ ID NO: 1.
2. The multimeric glycosylated hemoglobin monoclonal antibody of claim 1, wherein: the light chain variable region sequence is shown in SEQ ID NO: 2.
3. The multimeric glycosylated hemoglobin monoclonal antibody of claim 1, wherein: the heavy chain variable region sequence is shown in SEQ ID NO: 3.
4. A method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to any one of claims 1 to 3, comprising the steps of:
(1) Immunization of animals
Selecting female BALB/c mice of 6-8 weeks old, injecting the antigen into peripheral immune organs through blood circulation or lymph circulation, stimulating corresponding B lymphocyte clone, activating and proliferating the B lymphocyte clone, and differentiating the B lymphocyte clone into sensitized B lymphocyte;
(2) Cell fusion
By CO 2 Killing mice by gas, taking out spleens by aseptic operation, extruding and grinding in a plate, and preparing spleen cell suspension; mixing the prepared syngeneic myeloma cells with the spleen cells of the mice according to a certain proportion, and adding a fusogenic agent polyethylene glycol; under the action of polyethylene glycol, various lymphocytes and myeloma cells generateFusing to form hybridoma cells;
(3) Selective cultivation
Screening the fused hybridoma cells by adopting a HAT selective medium;
(4) Screening and cloning of hybridoma-positive clones
Cloning and culturing the hybridoma cells by adopting a limiting dilution method; screening out positive hybridoma cells capable of generating the required monoclonal antibody, and carrying out cloning amplification; through comprehensively identifying the type, subclass, specificity and affinity of the immunoglobulin of the secreted monoclonal antibody, identifying the epitope of the antigen and the molecular weight of the epitope, the monoclonal antibody is frozen in time;
(5) Monoclonal antibody purification
Purifying the ascites of the mice by using an ammonium sulfate salting-out method and a G protein chromatography method;
(6) The monoclonal antibodies are cross-linked.
5. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 4, wherein: the step (1) specifically comprises the following steps:
primary immunization, 50 ug/Ag, subcutaneous multipoint injection with Freund's complete adjuvant, 1.5mL, 3 weeks apart;
the second immunization, the dose route is the same as above, and Freund's incomplete adjuvant is added at intervals of 3 weeks;
the third immunization, the dose is the same, no adjuvant is added, the intraperitoneal injection is carried out, the blood is taken after 7 days to measure the titer, and the immunization effect is detected at intervals of 3 weeks;
boosting, 50ug of dosage, and intraperitoneal injection; after 3 days, spleen was taken for fusion.
6. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 5, wherein: the step (2) specifically comprises:
1) Preparation of feeder cells
BALB/c mice aged 6-10 weeks; pulling neck, killing, soaking in 75% alcohol, sterilizing for 3min, shearing skin with sterile scissors, and exposing peritoneum; injecting 6mL of culture solution into the tube, repeatedly flushing, and sucking out flushing liquid; putting into a 10mL centrifuge tube, and centrifuging at 1200rpm for 5min;
suspending with culture medium of 20% calf serum, and adjusting cell number to 1×10 5 /mL; add 96-well plates, 100 μl/well; culturing in incubator at 37deg.C;
2) Preparation of myeloma cells
Expanding and culturing myeloma cells 48-36 hours before fusion; on the day of fusion, lightly blowing down cells from the bottle wall by using an elbow dropper, and collecting the cells in a 50m centrifuge tube or a fusion tube; centrifuging at 1000r/min for 5-10 min, and discarding supernatant; adding 30mL of incomplete culture medium, and centrifugally washing once; then, re-suspending the cells in 10mL of incomplete culture medium, and uniformly mixing; taking myeloma cell suspension, adding 0.4% of a phenol blue dye solution as living cells for counting for later use;
3) Preparation of spleen cells
Taking an immunized BALB/c mouse, removing eyeballs, taking blood, and separating serum to be used as positive control serum in antibody detection; meanwhile, killing the mice through cervical dislocation, soaking in 75% alcohol for 5 minutes, lifting the skin at the left side abdomen after fixing on a culture dish, seeing spleen, changing ophthalmic scissors forceps, cutting peritoneum in an ultra clean bench by aseptic operation, taking out the spleen, placing the spleen in a dish which is filled with 10mL of incomplete culture medium, lightly washing, and carefully stripping off surrounding connective tissues; placing on a stainless steel screen in a plate, grinding into cell suspension by using a syringe needle core, and counting to enable spleen cells to enter an incomplete culture medium in the plate; blowing with a suction tube for several times to prepare single cell suspension; typically 1X 10 mice per mouse 8 -2.5×10 8 Individual spleen cells;
4) Cell fusion
Will be 1X 10 8 Spleen cells and 1X 10 7 Myeloma cell SP2/0 is mixed in a 50mL fusion tube, and incomplete culture medium is added to 30mL, and fully and uniformly mixed; centrifuging at 1000r/min for 5-10 min, and sucking the supernatant as much as possible; tapping the fusion tube bottom on the palm to loosen and uniformly deposit cells;
the preheated 50% PEG 1mL was added over 30s with a 1mL pipette with gentle agitation; sucking the suction tube and standing for 1min; adding the preheated incomplete culture solution, stopping PEG action, and continuously adding 1mL,2mL,3mL,4mL,5mL and 10mL in every 2min respectively; centrifuging at 800rpm for 5 minutes; discarding the supernatant;
adding 5mL of complete culture medium, gently blowing and sucking the precipitated cells, suspending and uniformly mixing the cells, and then supplementing the complete culture medium to 40-50mL; split charging 96-well cell culture plates, 100 μl per well, then placing the plates at 37deg.C, 5% CO 2 Culturing in an incubator; supplementing a selection medium after 6 hours; 50 mu L of each well, and half-changing the liquid with a selective medium after 3 days;
observing the growth condition of the hybridoma cells, and sucking out the supernatant for antibody detection when the hybridoma cells grow to more than 1/10 of the bottom area of the hole.
7. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 6, wherein: the step (3) is specifically that the antigen is diluted to 10ug/mL by coating liquid; adding 100 mu L/hole into the enzyme-labeled plate hole, and standing at 4 ℃ overnight or at 37 ℃ for adsorption for 2 hours; discarding the liquid in the hole, washing 3 times with washing liquid for 3 minutes each time, and drying; 100 mu L of sealing liquid is added to each hole to seal for 1 hour at 37 ℃; washing 3 times with washing liquid;
adding 100 mu L of hybridoma cell culture supernatant to be detected into each hole, and simultaneously setting positive control, negative control and blank control; incubation at 37 ℃ for 1 hour; washing and drying;
adding enzyme-labeled secondary antibody, incubating for 1 hour at 37 ℃ at 100 mu L per hole, washing, and drying;
adding substrate liquid, adding 100 mu L of freshly prepared substrate use liquid into each hole, and 20 minutes at 37 ℃;
in a ratio of 2mol/L H 2 SO 4 Terminating the reaction, and reading an OD value on an ELISA reader;
and (3) result judgment: P/N is equal to or greater than 2.1, or P is equal to or greater than N+3SD; if the negative control Kong Mose is or is near colorless, the positive control wells are clearly colored, and the results can be directly observed with the naked eye.
8. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 7, wherein: the step (4) is specifically that the spleen cells of the mice are prepared as feeder cells; preparing hybridoma cell suspension to be cloned, and diluting the hybridoma cell suspension to 3 different dilutions of 5, 10 and 20 cells per milliliter by using HT medium containing 20% serum;
added according to 5X 10 per milliliter 4 -1×10 5 Proportion of cells, respectively adding abdominal macrophages into the hybridoma cell suspension; split charging each hybridoma cell into one 96-well plate, wherein the amount of each well is 100 mu L;
37℃、5％CO 2 culturing for 6 days, and detecting antibodies after macroscopic cloning; observing under an inverted microscope, marking a hole in which only a single clone grows, and taking supernatant as an antibody for detection;
and (5) taking cells of the antibody detection positive hole, performing expansion culture, and freezing.
9. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 8, wherein: the step (5) is specifically to crosslink the staphylococcus protein A or the anti-mouse globulin antibody with a carrier by adopting an affinity purification method, prepare an affinity chromatographic column, combine the antibody and then elute.
10. The method for producing a multimeric glycosylated hemoglobin monoclonal antibody according to claim 9, wherein: in the step (6), the reaction system for coupling glycosylated hemoglobin monoclonal antibody is as follows:
antibody, concentration 2mg/mL,5mL;
glutaraldehyde, mass concentration 0.25%,200 μl;
centrifuging at 200rpm, and at 25 ℃ for 5 hours;
5mg/mL sodium borohydride was terminated.
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